Quantification of PepMV Viral Accumulation

Plant samples were ground in liquid nitrogen, and RNA was extracted using the NZY Total RNA Isolation kit (NZYTech, Portugal) following the manufacturer’s instructions. Extracts were treated with DNaseI (Sigma-Aldrich), and viral accumulation was quantified by RT-qPCR using the One-step NZYSpeedy RT-qPCR Green Kit and ROX Plus (NZYTech, Portugal) with specific primers for PepMV-EU and PepMV-CH2 (Gómez et al., 2009 (link)). To prepare viral RNA as pure as possible, virion purification was performed as in Agirrezabala et al. (2015) (link), and RNA extraction from virions was performed following the protocol described by AbouHaidar et al. (1998) (link). Then, 10-fold serial dilutions of the viral RNA from disassembled PepMV-EU or PepMV-CH2 virions were used to prepare the standard curves used for viral quantification.